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Biomedical subjects

G Ravagnan

Publications and source records attributed to G Ravagnan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cholesterol protects the phospholipid bilayer from oxidative damage.

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

Cholesterol↗
PubMed1995

Comparison of nephelometric and hemolytic techniques for determination of antistreptolysin O antibodies.

The sensitivity of a newly devised nephelometric method for determining antistreptolysin O antibodies was compared with the hemolytic inhibition assay. Three hundred-thirty single serum samples from children with and without evidence of group A streptococcal infection were analyzed by the two techniques. The nephelometric method results correlated well with those of the reference test (concordance: r = 0.88). Furthermore, 134 pairs of acute and convalescent phase sera from patients with culture-proven GAS infection and 50 pairs from children who served as control subjects were examined. The nephelometric assay was more sensitive in detecting significant ASO antibody rises than the hemolytic assay. The automated nephelometric method appears to be a much simpler and sensitive procedure for testing ASO antibodies.

Antibodies↗
PubMed1995

Relative value of selective group A streptococcal agar incubated under different atmospheres.

A commercially available selective group A streptococcal agar (ssA) was evaluated for the recovery of group A streptococci (GAS) in comparison with recovery from simultaneous cultures on conventional sheep blood agar (SBA). Both sets of plates were incubated in air, 5% CO2, and anaerobically for 48 h, with a first reading taken at 24 h. A total of 402 (67.0%) GAS were isolated from the 600 specimens that were submitted. Recovery of GAS was significantly greater after 48 h of incubation than after 24 h of incubation for each medium-atmosphere combination. After 48 h of incubation, the sensitivities of GAS detection obtained by each culture technique were as follows: ssA-anaerobic atmosphere, 98.5%; SBA-anaerobic atmosphere, 89.5%; ssA-CO2 atmosphere, 88.0%; SBA-air, 86.5%; SBA-CO2 atmosphere, 82.0%; and ssA-air, 74.6%. There were no cultures positive in air or CO2 which were not positive anaerobically on either medium. The increased sensitivity of detecting positive GAS cultures when incubation was done in an ssA-anaerobic atmosphere for 48 h uncovered patients truly infected with the organisms.

Aerobiosis↗
PubMed1994

Differential effects of short-term or prolonged cocaine exposure on peripheral blood cells in mice.

The effects of cocaine on the number of murine peripheral blood cells and on phenotypic expression of lymphocyte subsets were examined using different regimens of drug exposure. Cocaine administered to mice for 7 consecutive days at 1 and 10 mg/kg/day reduced the absolute number of circulating white blood cells and the relative number of cells stained as lymphocytes, polymorphonuclear cells and monocytes. Cocaine did not modify the relative proportion between lymphocytes, polymorphonuclear cells and monocytes. Parallely, there were no changes in the percentage of circulating CD4+ and CD8+ lymphocytes, while the number of natural killer cells increased. In sharp contrast, we didn't observe any significant change in peripheral blood cells after 30 days of consecutive drug administrations.

Animals↗
PubMed1994

Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence.

Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe.

The effect of cholesterol on the gel, the liquid-crystalline, and mixed phospholipid phases has been studied using the fluorescence properties of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Laurdan sensitivity to the polarity and to the dynamics of its environment reveals that cholesterol affects phospholipid bilayers in the gel phase by expelling water and by increasing the amount of dipolar relaxation. In the liquid-crystalline phase, the effect of cholesterol is a reduction of both water concentration and amount of dipolar relaxation. Detailed studies of Laurdan excitation and emission spectral contours as a function of cholesterol concentration show that there are some cholesterol concentrations at which Laurdan spectral properties changes discontinuously. These peculiar cholesterol concentrations are in agreement with recent observations of other workers showing the formation of local order in the liquid-crystalline phase of phospholipids upon addition of phospholipid derivatives of pyrene. A local organization of phospholipids around cholesterol molecule seems to be produced by the presence of peculiar concentrations of cholesterol itself. This local organization is stable enough to be observed during the excited state lifetime of Laurdan of approximately 5-6 ns.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation.

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Lipid Bilayers↗
PubMed1994

Suppression of rat epididymal sperm immunogenicity by a seminal vesicle secretory protein and transglutaminase both in vivo and in vitro.

The pretreatment of epididymal spermatozoa with SV-IV, one of the major secretory protein produced by the epithelium of adult rat seminal vesicles, was found to markedly decrease their ability to induce in vivo peritoneal macrophage activation, measured as class II major histocompatibility complex surface antigen expression, superoxide anion production, phagocytic activity, and antigen presentation. In addition, the treatment of spermatozoa with SV-IV produced a significant decrease of their immunogenicity evaluated in vitro by [3H]thymidine incorporation in splenocyte/spermatozoon co-culture. The concurrent presence of SV-IV and transglutaminase, an enzyme secreted in large amounts from the rat anterior prostate, amplified these phenomena. The suppression of the epididymal sperm immunogenicity is suggested to be of crucial importance for the prevention of the immune response to the sperm introduced in the immunocompetent female genital tract during coitus.

Animals↗
PubMed1993

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

2-Naphthylamine↗
PubMed1993

EBV membrane receptor (CR2) is phosphorylated by protein kinase C (PKC) in the early stages of virus entry into lymphoblastoid cells line (Raji).

Labeling the EBV membrane with octadecylrhodamine-b-chloride (R18) we were able to monitor spectrofluorometrically the early events of EBV fusion, under conditions in which we could affect PKC activity. Binding of EBV to Raji cells induces PKC translocation from the cytosol to the plasma membrane and 32P incorporation into its cellular receptor CR2. CR2 phosphorylation is completely inhibited when cells are preincubated with the PKC inhibitor calphostin c. This treatment also generates a strong inhibition of EBV fusion. Taken together this result suggests a key role of CR2 phosphorylation in the EBV entry into Raji cells.

Adenosine Triphosphate↗
PubMed1993

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole.

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.

Base Sequence↗
PubMed1993

Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV.

SV-IV, a 9.8-kd protein isolated from rat seminal vesicle secretion, has been shown to have strong non-species-specific immunosuppressive, anti-inflammatory, antithrombotic, and antiphospholipase A2 properties. The present investigation was undertaken to determine the mechanism of action of its immunosuppressive effects. It was found that SV-IV is a potent inhibitor of interleukin-1 (IL-1) release from lipopolysaccharide (LPS)-activated human adherent monocytes and an effective inhibitor of IL-1-induced thymocyte proliferation. The ability of SV-IV to form a noncovalent dimeric association with IL-1 alpha but not with IL-1 beta, its ability to induce a marked decrease of IL-1 binding to its own receptors on the thymocyte surface, and its capacity to bind specifically to the macrophage plasma membrane might play an important role in the molecular mechanism of these inhibitory effects.

Animals↗
PubMed1993

Susceptibility of human liver cell cultures to hepatitis C virus infection.

To develop a cell culture system susceptible to infection by hepatitis C virus (HCV), human fetal hepatocytes, grown in serum-free medium, were inoculated with serum samples from two HCV-infected patients. Viral RNA sequences were detected by polymerase chain reaction, using primers specific for the 5' noncoding region of HCV, in extracts prepared from the hepatocyte cultures as early as 5 days after inoculation. Virus was also released from the infected cells into the medium. The HCV strains could be serially passaged three times into fresh liver cell cultures using intracellular virus as inoculum. Evidence that HCV replication really took place in primary human fetal hepatocytes was also obtained by detection of minus-strand viral RNA (replication intermediate) in cell extracts and of viral antigens in the infected cells.

Base Sequence↗
PubMed1993

Effect of synthetic thymic hormones on the cocaine-induced inhibition of the primary immune response in mice.

The effects of thymosin alpha 1 (T alpha 1) and thymopentin (TP5) on the cocaine-induced impairment of the primary antibody response to sheep red blood cells (SRBC) was studied. The administration of cocaine from day -4 to the day of immunization (day 0) induced a significant impairment of the response to the T-dependent antigen SRBC, as evaluated on day 5 post-immunization by the Splenocyte-Induced SRBC Hemolysis (SIH) assay. The analysis of the responses to immunogen elicited from each single mouse indicated that, under the experimental conditions used, cocaine acted by exerting more an "all or nothing" effect rather than by modulating the strength of the immune response. Both T alpha 1 and TP5, injected into mice during cocaine administration and for 4 days after, induced a significant recovery of the response to SRBC. Our experiments did not show any great differences in the overall efficacy of the two drugs, although they showed quite a different dose-response effect. The results of the present investigation demonstrated the capability of TP5 and T alpha 1 to reverse the cocaine-induced impairment of the response to SRBC and suggested that the effect of the two peptides may be related to their immunomodulating activities on T-cell functions.

Animals↗
PubMed1993

Yersinia pseudotuberculosis in Italy. Attempted recovery from 37,666 samples.

From 1981 to 1991, 37,666 human, animal, food and environmental samples were cultured for Yersinia pseudotuberculosis using direct plating methods and/or cold enhancement techniques. Despite an intensive surveillance and adequate culture methods, Y. pseudotuberculosis was isolated from stools of 0.05% (5/9,720) of patients with acute enteritis, and alimentary tracts of 0.1% (10/6,849) of apparently healthy animals. No Y. pseudotuberculosis strains were recovered from stools of 4,726 health controls nor from the appendices (656), mesenteric lymph nodes (84), and stools (421) of 656 patients operated for suspected appendicitis. Of the 10,842 food and 4,368 environmental samples, none yielded positive cultures for Y. pseudotuberculosis.

Animals↗
PubMed1993

Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence.

Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl- and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Use of octadecylrhodamine fluorescence dequenching to study vesicular stomatitis virus fusion with human aged red blood cells.

Human erythrocytes were separated into five fractions representing different age groups. In each group phospholipid inside-outside translocation was determined by quantitation of the amino phospholipids phosphatidylserine and phosphatidylethanolamine and their lyso-derivatives by thin layer chromatography. To assess the role of transbilayer phospholipid distribution in the recognition and fusion of vesicular stomatitis virus (VSV) and human aged erythrocytes, we monitored the fusion kinetics using the octadecylrhodamine dequenching assay. Fusion of VSV with each single group of red blood cells (RBC) was not detectable with the youngest cells (F1 group) but increased with RBC aging (F2-F5 groups). The same increase in fusion was observed with microvesicles generated from RBC in which aging was mimicked by incubating the cells with Ca2+ in the presence of the Ca2+ ionophore A23187. Conversion of the aminophospholipids to the trinitrophenyl derivative by reaction with trinitrobenzensulfonate completely inhibits fusion on ghosts in which aging was artificially induced by translocation of aminophospholipids in the outer leaflet (symmetric ghosts). These results indicate that RBC become susceptible to VSV fusion during aging and in all pathology related to the aging process.

Erythrocyte Aging↗